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Uvod. Traheokutana fistula, patološki spoj dušnika i kože predstavlja jednu od kasnih komplikacija traheotomije. Prema podacima iz literature, incidencija traheokutane fistule je preko 40% u pedijatrijskih pacijenata. Do sada su opisane brojne kirurške tehnike zbrinjavanja traheokutane fistule, ali još uvijek nije pronađena tehnika i način zbrinjavanja koji bi predstavljao zlatni standard. Kirurško zbrinjavanje fistula sa sobom nosi i moguće komplikacije stoga je poželjno pronaći novo, bolje rješenje. Pentadekapeptid BPC 157 je izoliran iz humanog želučanog soka, a karakterizira ga stabilnost pri neutralnom pH i što za svoje djelovanje ne mora koristiti nosač što ga razlikuje od drugih peptida.U dosadašnjim istraživanjima nije utvrđena letalna doza što ga čini sigurnim za primjenu. BPC 157 pokazao je svoju djelotvornost u cijeljenju rana i fistula općenito stoga je poželjno utvrditi njegov učinak na modelu traheokutane fistule u štakora. ----- Materijali i metode. Mužjaci Wistar albino štakora, tjelesne mase 220-280 g podvrgnuti su standardnoj horizontalnoj traheotomiji prilikom koje je kreirana traheokutana fistula veličine 4 mm. Životinje su podijeljene u skupine: BPC 157 μg/ng, peroralna/intraperitonejska primjena, kontrolna skupina, L-NAME (i.p.), L-arginin (i.p.), L-NAME+ L- arginin (i.p.), L-NAME+ BPC 157 μg/ng( i.p.), L-NAME+ L- arginin+ BPC 157 μg/ng (i.p.), L- arginin + BPC 157 μg/ng (i.p.). Učinak ispitivanih agensa na proces cijeljenja promatran je kroz tri vremenska intervala- treći, peti i sedmi postoperacijski dan. Sedmi postoperacijski dan nastupilo je žrtvovanje životinja i vrednovanje rezultata. ----- Rezultati. BPC 157 doveo je do poboljšanja procesa cijeljenja traheokutane fistule kroz sve ispitivane vremenske intervale što je u konačnici rezultiralo gotovo potpunim zatvaranjem fistule. Njegov učinak je bio nepromijenjen neovisno o primjenjenoj dozi, 10μg/kg - 10ng/kg, kao i načinu primjene (p.o./i.p.). Uspješno je poništavao nepovoljni učinak L- NAME, a u zajedničkoj primjeni s L- NAME i L- argininom je pokazao rezultat sličan onome prilikom samostalne primjene. L- arginin je sedmi postoperacijski dan pokazao statitički značajan rezultat. Sve navedeno govori u prilog interakcije BPC 157 i NO sustava u ovome modelu. ----- Zaključak. BPC 157 ubrzava cijeljenje traheokutane fistule u štakora. Svoje djelovanje ostvaruje zahvaljujući interakciji s NO sustavom., Introduction. One of the common late complications of a tracheotomy is tracheocutaneous fistula. The surgical procedure is needed to close such fistulas. BPC 157 represents an anti-ulcer peptide (GEPPPGKPADDAGLV, M.W. 1419) successful in trials for inflammatory bowel disease, wound treatment, effective alone without a carrier. BPC 157 interferes with NO-system in vitro/in vivo on different models and different animal species. The impact of BPC 157 and NO-system on the gastrocutaneous, colocutaneous, and oesophagocutaneous fistulas healing process indicates the potential effect of BPC 157 in the tracheocutaneous fistula healing process, which has not yet been investigated. ----- Matherials and methods. Albino Wistar male rats were used in this experiment, 220-280 g. Tracheocutaneous fistula, 4 mm trachea and skin defect, was surgically made under anaesthesia. Animals were treated according to the experiment protocol: (a)) drinking water p.o., (b)) BPC 157 (10 μg/kg, 12 ml/rat/day) p.o., (c)) BPC 157 (10 ng/kg, 12 ml/rat/day) p.o., (d)) saline 5ml/kg/day i.p., (e)) BPC 157 (10μg/kg, 5ml/kg/day) i.p., (f)) BPC 157 (10 ng/kg, 5 ml/kg/day) i.p., (g)) L-NAME (5 mg/kg/day) i.p., (h)), L- arginine (100 mg/kg/day) i.p., (i)) L-NAME L- arginine i.p., (j)) L- NAME BPC 157 (μg) i.p., (k)) L- arginine BPC 157 (μg) i.p., (l)) L-NAME L- arginine BPC 157 (μg) i.p., (m)) L-NAME BPC 157 (ng) i.p., (n)) L- arginine BPC 157 (ng) i.p., (o)) L-NAME L- arginine BPC 157 (ng) i.p.. Seventh postoperative day animals were euthanized. Fistula specimens were harvested and macroscopic and histological analysis was made. ----- Results. A consistent counteracting beneficial effect was shown in all animals treated with BPC 157, alone and with (L-NAME) and/or L-arginine in a 7-day interval. BPC 157 accelerated the healing of tracheocutaneous fistulas and showed macroscopic and histological healing improvements. The tracheocutaneous fistulas healing was improved speedily (BPC 157 completely counteracted L-NAME effects (L-NAME+BPC 157 and L-NAME+L-arginine+ BPC 157 groups), or with delay and to less extent (L-arginine) or aggravated, rapidly and prominently (L-NAME). L-arginine reduces aggravation by NOS-blockade (L-NAME) to the control level. Also, BPC 157 more than nullifies the effect of L-NAME. ----- Conclusion. BPC 157 improved the healing of both tracheal and skin defects and mediated fistula closing. This effect was shared by L-arginine, with an opposite effect seen by L-NAME, thereby portraying BPC 157/NO-system involvement in the healing of tracheocutaneous fistula.

Atrofija mišića je neminovna pri imobilizaciji ekstremiteta. Definitivno smanjuje izglede za dobrim ishodom liječenja. Radi se uglavnom o reverzibilnom oštećenju mišića koje u većini slučajeva rezultira samo produženom rehabilitacijom, međutim, nije rijedak slučaj da dovodi do trajne smanjene funkcije ekstremiteta. S obzirom da ne postoji za sada farmakoterapija koja bi spriječila ili umanjila razvoj atrofije mišića mi smo postavili hipotezu da pentadekapeptid BCP157 preko NO sustava sprečava razvoj atrofije te kasnije pospješuje regeneraciju mišića, imajući u vidu ranija istraživanja o resekciji i natučenju mišića, resekciji tetive i ligamenta, cijeljenju rana te protuupalnom djelavnju pentadekapeptida BCP 157. Štakore sa reseciranim patelarnim ligamentom na stražnjoj lijevoj nozi smo tretirali peroralno i parenteralno s BPC157 (10ug/kg), L-NAME (5 mg/kg) i L-argininom (100 mg/kg) pojedinačno i/ili u kombinaciji. Imajući u vidu da kod reseciranog patelarnog ligamenta dolazi do atrofije mišića, a shodno prethodnim našim radovima, mjerili smo učinak pentadekapeptida BPC 157 tako što smo činili kliničko-funkcionalne testove i histološki analizirali promjene u mišiću. Pomoću RT-PCR-a su istraživane razine eNOS, iNOS, COX-2 mRNA iz tkiva mišića. Doprinos rada (definiranje opsežnosti razvoja atrofije te sprečavanje iste, te benefitni učinak BPC 157) predstavljat će značajnu znastvenu novost. Muscle atrophy is inevitable in limb immobilization. Definitely reduces the chances of a good outcome. It is usually about reversible muscle damage, which in most cases results in only prolonged rehabilitation, however, is not a rare case that leads to a permanently reduced extremity function. Given that there is no pharmacotherapy that would prevent or reduce the development of muscle atrophy, we hypothesized that pentadecapeptide BCP157 in relation to NO system reduces the development of atrophy and subsequently promotes muscle regeneration, taking into account previous studies of muscle resection, tendon and ligament resection, wound healing and favorable antiinflammatory effects of pentadecapeptide BCP 157. Rats with a resected patellar ligament on the back left leg will be treated with oral and parenteral BPC157 (10ug/kg, 10ng/kg), L-NAME (5 mg / kg) and L-arginine (100 mg / kg) alone and / or in combination. Having in mind that the resection of patellar ligament causes muscle atrophy, according to our previous work, we will measure the effects of pentadecapeptide BPC 157, we will do clinical functional tests and histological analysis of the lesions in the muscle. RT-PCR will investigated levels of eNOS, iNOS, COX-2 mRNA from muscle tissue. The contribution of study will be in defining comprehensive development of atrophy and prevention of the same, and benefit effects of BPC 157 which will provide a significant scientific novelty.

Svakodnevno, a posebno tijekom sportskih aktivnosti česta su oštećenja mišića i tetiva. Mišićno-tetivni spoj je najslabija komponenta u cijeloj jedinici i veliki broj oštećenja dogaĎa se na njemu. Na temelju rezultata ranijih istraţivanja u kojima terapija BPC 157 dovodi do cijeljenja oštećene tetive ili mišića, postavili smo hipotezu da BPC 157 utječe pozitivno na cijeljenje i ponovnu uspostavu čvrste i kvalitetetne mišićno-tetivno sveze mišića kvadricepsa u štakora. Eksperimentalnim ţivotinjama, s kirurški razdvojenim mišićnim od tetivnih niti u području četveroglavog mišića desne natkoljenice raspodjeljenim u 3 skupine davali smo: BPC 157 u dozi od 10 ng/kg, BPC 157 u dozi od 10 μg/kg ili fiziološku otopinu. Pratili smo kliničko-fiziološke promjene, a po ţrtvovanju napravili makroskopske i histološke analize te biomehanička i laboratorijska testiranja tkiva. U patohistološkim preparatima obje skupine BPC tretiranih ţivotinja u odnosu na kontrolnu, bila je značajno manja upala te puno brţa i kvalitetnija reparacija mišićno-tetivog spoja. Cijeljenje je bilo popraćeno većom ekspresijom eNOS mRNA i smanjenom razinom NO i slobodnih radikala kod jedinki koje su tretirane BPC 157 u odnosu na kontrolnu skupinu. Isto je rezultiralo manjim razvojem atrofije mišića i nastajanja defekta na mjestu spoja. Tretirane ţivotinje bolje su hodale, imale jaču snagu pritiska i nisu razvijale kontrakturu noge. Naši rezultati pokazuju da bi se BPC 157 mogao koristiti kod ozljeda mišićno-tetivne sveze obzirom da poboljšava cijeljenje, a za kliničku praksu neophodna su još dodatna istrazivanja. Daily and especially during sports activities muscle and tendon are often damaged. How is the muscular tendon junction weakest component in the entire unit so that a large number of defects occurring right on that part. According to the results of previous studies in which therapy BPC 157 leads to successful healing of injuries isolated tendon or muscle tissue, we hypothesized that BCP157 positively affects on the healing and restoration of solid and quality muscular-tendon connection. Experimental animals with surgical separate muscle from tendon in the right muscle quadriceps and randomly devided into the 3 groups, will be treated parenterally with: BPC 157 at a dose of 10 ng/kg, BPC 157 at a dose of 10 μg/kg or saline. During research, we followed clinical-physiological changes and after sacrificing macroscopic, histological and biomechanical tissue testing were done. In the pathohistological preparations of both groups of BPC treated animals in comparison to the control, there was noticeably less inflammation and a much faster and better repair of the muscle-tendon join. Healing was followed with greater expression of the eNOS mRNA and reduced levels of NO and free radicals. This has resulted, macroscopically, with the lower development of muscle atrophy and the formation of a defect in the muscle-tendon junction. Treated animals walked better, they had stronger pressure with leg and the contractures did not developed. The contribution of labor would represent unquestionable novelty in healing muscle-tendon junction.

We revealed an immediate and hours-lasting particular NO-specific parallel miotic effect of L-NAME and L-arginine in rats and guinea pigs and a stable gastric pentadecapeptide BPC 157 157-particular effect versus that of atropine-induced mydriasis while examining the NO system role in the normal pupils responses and pupils with atropine-induced mydriasis. We also assessed the responses to BPC 157 and its possible modulation of the changes caused by L-NAME/L-arginine and atropine. We administered locally (two drops/eye) or systemically (intraperitoneally/kg) [BPC 157 (0.4µg/eye ; 10µg, 10ng, 10pg/kg), L-NAME (0.1mg/eye ; 5mg/kg), and L-arginine (2mg/eye ; 100mg/kg) alone and combined] at 3 min prior to assessment (normal pupils) or alternatively at maximal 1% atropine-induced mydriasis (30 min after two drops were administered to each eye). L-NAME/L-arginine. Normal pupil. L-NAME-miosis and L-arginine-miosis shortened and attenuated each other’s responses when combined (L-NAME+L-arginine) (except with guinea pigs treated locally) and were thereby NO-specific. Atropine-pupil. Both L-NAME and L-arginine counteracted atropine-induced mydriasis. With few exceptions, the atropine+L-NAME+L-arginine-animals showed a consistent shift toward the left. BPC 157. Normal pupil. Always, BPC 157 alone (both species ; locally ; systemically ; all regimens) did not affect normal pupils. Despite specific exceptions, BPC 157 distinctively affects L-arginine-miosis (prolongation) and L-NAME-miosis (shortening). When L-arginine and L-NAME were combined (L-NAME+L-arginine+BPC 157), the effect was less pronounced. Atropine-pupil. BPC 157 alone counteracted atropine-induced mydriasis. With few exceptions (when administered with L-NAME or L-arginine or L-NAME+L-arginine), BPC 157 augments their counteracting effects. Thus, along with its L-NAME/L-arginine effects, BPC 157 participates in ocular control, potentially via NO-mediated and cholinergic mechanisms.

Prethodna istraživanja pokazala su da BPC 157 sprječava nastanak okluzivnog tromba i ubrzava razgradnju već stvorenog ugruška nakon formiranja anastomoze aorte, a da, s druge strane, smanjuje vrijeme krvarenja kod štakora tretiranih varfarinom, heparinom i aspirinom. Temeljem toga, nametnula se potreba određivanja njegova utjecaja na agregaciju trombocita i viskoelastična svojstva krvnog ugruška. Za ispitivanje eventualne uključenosti NO sustava u djelovanje BPC 157 korišten je selektivni inhibitor topljive gvanilil ciklaze, ODQ. Životinje (n=60) su najprije podijeljene u skupine ovisno o tome koji antiagregacijski lijek primaju (1.aspirin, 2.klopidogrel, 3.cilostazol), a te skupine su tada nasumično podijeljene u četiri podskupine koje su primale a) fiziološku otopinu, b) BPC 157, c) ODQ, d) BPC 157 + ODQ. Mjerenja su izvršena impendancijskom agregometrijom s 4 različita agonista (ADP, AA, AA/PGE1, kolagen), te rotacijskom tromboelastometrijom s 3 različita agonista (preko vanjskog i unutarnjeg puta zgrušavanja, te bez doprinosa trombocita). Iz rezultata dobivenih usporedbom podskupina a i b, te b i d unutar svake skupine možemo zaključiti da BPC 157 oporavlja inhibiranu agregaciju trombocita, ali da nema utjecaja na viskoelastična svojstva krvnog ugruška u ispitivanih štakora. As a natural extension of previous research that confirmed the role of BPC 157 in the prevention of obstructive thrombus formation and rapid destruction of an already formed one after aortic anastomosis, but also shortening of the bleeding time in rats treated with anticoagulants and aspirin, there was a necessity to determine how BPC 157 influences platelet aggregation and viscoelastic properties of the blood clot. To assess its relation to NO system, sCG selective inhibitor (ODQ) was used. Rats (n=60) were divided into groups depending on the antiaggregatory drug they were treated with (1.aspirin, 2.clopidogrel, 3.cilostazol). Groups were further divided into four subgroups treated with a) normal saline, b) BPC 157, c) ODQ, d) BPC 157 + ODQ. Impendance aggregomery measurements with four agonists (ADP, AA, AA/PGE1 and collagen), and also rotational thromboelastometric measurements with 3 agonists (for initiating external and internal coagulation pathway and without platelet contribution) were performed. Based on the results obtained by comparing subgroups a versus b, and b versus d in each group, we can conclude that BPC 157 rescues inhibited platelet aggregation, but it has no effect on viscoelastic properties of the blood clot.

Stable gastric pentadecapeptide BPC 157 was previously used to ameliorate wound healing following major surgery and counteract diclofenac toxicity. To resolve the increasing early risks following major massive small bowel resectioning surgery, diclofenac combined with nitric oxide (NO) system blockade was used, suggesting therapy with BPC 157 and the nitric oxide synthase (NOS substrate) L-arginine, is efficacious. Immediately after anastomosis creation, short-bowel rats were untreated or administered intraperitoneal diclofenac (12 mg/kg), BPC 157 (10 μg/kg or 10 ng/kg), L-NG- nitroarginine methyl ester (L-NAME, 5 mg/kg), L-arginine (100 mg/kg) alone or combined, and assessed 24 h later. Short-bowel rats exhibited poor anastomosis healing, failed intestine adaptation, and gastrointestinal, liver, and brain lesions, which worsened with diclofenac. This was gradually ameliorated by immediate therapy with BPC 157 and L-arginine. Contrastingly, NOS-blocker L-NAME induced further aggravation and lesions gradually worsened. Specifically, rats with surgery alone exhibited mild stomach/duodenum lesions, considerable liver lesions, and severe cerebral/hippocampal lesions while those also administered diclofenac showed widespread severe lesions in the gastrointestinal tract, liver, cerebellar nuclear/Purkinje cells, and cerebrum/hippocampus. Rats subjected to surgery, diclofenac, and L-NAME exhibited the mentioned lesions, worsening anastomosis, and macro/microscopical necrosis. Thus, rats subjected to surgery alone showed evidence of deterioration. Furtheremore, rats subjected to surgery and administered diclofenac showed worse symptoms, than the rats subjected to surgery alone did. Rats subjected to surgery combined with diclofenac and L-NAME showed the worst deterioration. Rats subjected to surgery exhibited habitual adaptation of the remaining small intestine, which was markedly reversed in rats subjected to surgery and diclofenac, and those with surgery, diclofenac, and L-NAME. BPC 157 completely ameliorated symptoms in massive intestinal resection-, massive intestinal resection plus diclofenac-, and massive intestinal resection plus diclofenac plus L- NAME-treated short bowel rats that presented with cyclooxygenase (COX)-NO-system inhibition. L-arginine ameliorated only L-NAME-induced aggravation of symptoms in rats subjected to massive intestinal resection and administered diclofenac plus L-NAME.

Cannabidiol, the major non-psychoactive component of marijuana, has various pharmacological actions of clinical interest. It is reportedly effective as an anti-inflammatory and anti-arthritic in murine collagen-induced arthritis. The present study examined the anti-inflammatory and anti-hyperalgesic effects of cannabidiol, administered orally (5-40 mg/kg) once a day for 3 days after the onset of acute inflammation induced by intraplantar injection of 0.1 ml carrageenan (1% w/v in saline) in the rat. At the end of the treatment prostaglandin E2 (PGE2) was assayed in the plasma, and cyclooxygenase (COX) activity, production of nitric oxide (NO; nitrite/nitrate content), and of other oxygen-derived free radicals (malondialdehyde) in inflamed paw tissues. All these markers were significantly increased following carrageenan. Thermal hyperalgesia, induced by carrageenan and assessed by the plantar test, lasted 7 h. Cannabidiol had a time- and dose-dependent anti-hyperalgesic effect after a single injection. Edema following carrageenan peaked at 3 h and lasted 72 h; a single dose of cannabidiol reduced edema in a dose-dependent fashion and subsequent daily doses caused further time- and dose-related reductions. There were decreases in PGE2 plasma levels, tissue COX activity, production of oxygen-derived free radicals, and NO after three doses of cannabidiol. The effect on NO seemed to depend on a lower expression of the endothelial isoform of NO synthase. In conclusion, oral cannabidiol has a beneficial action on two symptoms of established inflammation: edema and hyperalgesia.

Zusammenfassung Das Renin-Angiotensin-Aldosteron-System und das NO-System sind zwei bedeutende Komponenten der Blutdruckregulation, die in enger Verbindung miteinander stehen. Ausgehend von der Hypothese einer Beteiligung von NO an der Beeinflussung des AT1-Rezeptors durch den AT2-Rezeptor wurden komplexe physiologische Untersuchungen der Herz- und Nierenfunktion, zwei wichtigen Stellgliedern in der Blutdruckregulation, an AT2-Rezeptor-defizienten Mäusen durchgeführt. In diesem Tiermodell ist der AT1-Rezeptor hochgeregelt und bietet somit die Möglichkeit, die Funktion dieser Rezeptorisoform ohne begleitende Effekte des AT2-Rezeptors zu untersuchen. Zusätzlich wurde das NO- System durch Gabe von L-NAME supprimiert und mit DOCA-Salz stimuliert. Für das bessere Verständnis systemischer Vorgänge wurden auch molekularbiologische und histologische Befunde herangezogen. Sowohl unter L-NAME als auch DOCA-Salz kam es zu deutlichen Veränderungen der Nierenfunktion. Der Einfluß auf die Funktion des Herzen war deutlich geringer. Dieser Befund unterstreicht somit die besondere Bedeutung der Niere für die Langzeitregulation des Blutdruckes. Die L-NAME-Hypertonie wird maßgeblich durch die Wirkung des AT1-Rezeptors bestimmt. Sowohl die NO-Blockade als auch der Knockout des AT2-Rezeptors erwiesen sich dabei als starke Stimuli für nierenphysiologische Veränderungen. Die stärkeren renalen Effekte sowie die ausgeprägtere linksventrikuläre Hypertrophie in KO-Mäusen bestätigen die antagonistische Wirkung des AT2-Rezeptors auf den AT1-Rezeptor. Dabei zeigt sich vor dem Hintergrund der AT1-Rezeptor-Genexpressionsanalyse kein Anhaltspunkt für eine NO-vermittelte Beeinflussung des AT1-Rezeptors durch den AT2-Rezeptor. Auch der DOCA-Salz-Hypertonus stellt einen Widerstandshochdruck dar, der allerdings nicht primär vom AT1-Rezeptor bestimmt wird. Es konnte gezeigt werden, daß sowohl DOCA-Salz als auch der Knockout des AT2-Rezeptors starke Stimuli für die renale iNO-Synthese sind. Die pharmakologische Blockade von iNOS zeigte allerdings, daß dieses Verhalten hämodynamisch nicht von Bedeutung ist. Die Erfassung der Herz-Kreislauffunktion in L-NAME und DOCA-Salz behandelten AT2-/y zeigte im Zusammenhang mit Genexpressionsanalysen des AT1-Rezeptors entgegen der Anfangshypothese keine NO-vermittelte Beeinflussung des AT1-Rezeptors durch den AT2-Rezeptor., Summary The Renin Angiotensin Aldosteron System and the NO system are two important components of the blood pressure regulation that are closely intertwined. It is hypothesized that the AT1 receptor is influenced by the AT2 receptor in participation with NO.To test this hypothesis, complex physiological investigations of the heart and kidney were performed on AT2 receptor- deficient mice. In this animal model, the AT1 receptor is upregulated and provides the opportunity to examine the function of the AT1 receptor without concomittant effects of the AT2 receptor. Additionally, the NO system was suppressed by L-NAME and stimulated with DOCA salt respectively. For a better understanding of systemic processes, the physiological results were taken into consideration with both gene expression analyses and histological findings. Both L-NAME and DOCA had strong effects on kidney function. The influence on heart function was much weaker. These findings underline the dominat role of the kidney in long-term blood pressure regulation. L-Name hypertension is mainly caused by AT1 receptor effects. Both the NO blockade and the knockout of the AT2 receptor were strong stimuli for alterations in kidney function. The stronger renal effects, as well as the more pronounced left ventricular hypertrophy in AT2-/y, underline the antagonistic effects of the AT2 receptor on the AT1 receptor. The gene expression analysis of the AT1 receptor does not support the view, that the AT1 receptor is influenced by the AT2 receptor acting on NO. The blood pressure increase in DOCA-salt treated mice is also caused by an increase in total peripheral resistance, that is not primarily determined by the AT1 receptor. It has been shown that both DOCA-salt and the knockout of the AT2 receptor are strong stimuli for the renal iNO synthesis. The pharmacological blockade of iNOS showed no hemodynamical relevance. The analysis of kidney and heart function, together with gene expression analyses and histological investigations, showed no effect on the AT1 receptor by the AT2 receptor via NO. The initial hypothesis was rejected.

Nitric oxide synthase expression in AT 2 receptor–deficient mice after DOCA-salt. Background Angiotensin II type 2 receptor–deficient mice (AT 2 -/y) provide an opportunity to study the relationship between the angiotensin II type 1 receptor (AT 1 ) and nitric oxide synthase (NOS) isoforms without concomitant AT 2 receptor–related effects. To test this relationship, the expression of renal NOS isoforms (neural, inducible, and endothelial) in AT 2 -/y and AT 2 +/y mice was examined. The mice were challenged with deoxycorticosterone acetate (DOCA)-salt to stimulate NO generation. Methods Gene expression analyses by real-time polymerase chain reaction (PCR) (TaqMan) were performed in kidneys to characterize neuronal nitric oxide synthase (nNOS), epithelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), and the AT 1 receptor. Pressure-natriuresis experiments were done to determine the physiologic background. Results AT 2 -/y mice showed nNOS and iNOS up-regulation. DOCA-salt increased iNOS expression more in AT 2 -/y mice than in AT 2 +/y mice. Immunohistochemistry localized the iNOS expression with DOCA-salt mainly in the glomeruli. eNOS was not different between the groups, and was not affected by DOCA-salt. DOCA-salt increased mean arterial pressure more in AT 2 -/y mice than in AT 2 +/y mice. Concomitantly, the pressure-natriuresis relationship was shifted to the right in AT 2 -/y and AT 2 +/y mice after DOCA-salt. DOCA-salt decreased renal blood flow (RBF) and glomerular filtration rate (GFR) in both groups. iNOS blockade did not lower blood pressure. Conclusion We conclude that AT 2 receptor deletion and concomitant up-regulation of the AT 1 receptor is associated with up-regulation of nNOS and iNOS. Under DOCA-salt, renal iNOS expression was further increased. Because iNOS inhibition did not change blood pressure, iNOS may not be involved in the hemodynamics, but may contribute to organ damage.

Nitric oxide (NO)­containing neurons are widely distributed within the central nervous system, including regions involved in the control of reproduction and sexual behavior. Nitrergic neurons may co-localize with gonadal hormone receptors and gonadal hormones may influence neuronal NO synthase expression in adulhood as well as during development. In rodents, the female, in physiological conditions, is exposed to short­term changes of gonadal hormones levels (estrous cycle). Our studies, performed in mouse hypothalamic and limbic systems, reveal that the expression of neuronal NO synthase may vary according to the rapid variations of hormonal levels that take place during the estrous cycle. This is in accordance with the hypothesis that gonadal hormone activation of NO-cGMP pathway is important for mating behavior. NO-producing system appears particularly sensitive to alterations of endocrine balance during development, as demonstrated by our experiments utilizing perinatal exposure to bisphenol A, an endocrine disrupting chemical. In fact, significant effects were detected in adulthood in the medial preoptic nucleus and in the ventromedial subdivision of the bed nucleus of the stria terminalis. Therefore, alteration of the neuronal NO synthase expression may be one of the causes of the important behavioral alterations observed in bisphenol-exposed animals., Italian Journal of Anatomy and Embryology, Vol 115, No 1/2 (2010)